CD147, Soluble(EMMPRIN), ELISA Kit

Product#: KG573
$1,246.00
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CD147, Soluble(EMMPRIN), ELISA Kit

Cat. No. KG573
Store at -20°C
Size 1kit

Description 

Sensitivity (the corresponding concentration of blank absorbance + 2SD); 0.30 ng/Ml.

CD147, a member of the immunoglobulin superfamily, is a transmembrane glycoprotein included two immunoglobulin-like domains. CD147 is also known as extracellular matrix metalloproteinase inducer (EMMPRIN) or basigin and is enriched on the surface of many malignant tumor cells and stromal cells. The up-regulation of the matrix metalloproteinases (MMPs) by CD147 is believed to be progression of malignancies through tumor growth, invasion and metastasis.

In the recent studies, EMMPRIN fragment (22KDa) is released in a soluble form by the protelytic cleavage of MMPs from tumor cell surface. There are several evidences that the soluble CD147 is existed into the culture medium of several tumor cells.

Test Principle

This assay kit employs the quantitative sandwich ELISA technique based on two mouse monoclonal antibodies against to specific different epitope of EMMPRIN. One of specific antibody is pre-coated onto 96-well microplate.

Calibrators and samples (culture medium and clinical sample, e.g. urine) are applied into the wells and incubated, EMMPRIN is captured by the coated antibody. Following, the microplate is incubated with biotinylated antibody. And next step, HRP-conjugated streptavidin is added to the well and incubated. Finally, a chromogenic substrate solution (H2O2 and OPD) is applied to each test well and color develops in proportion to
EMMPRIN level. The enzymatic reaction is stopped by addition of sulphuric acid and absorbance of the color intensity is measured spectrophotometrically at 490 nm. Calibration curve is constructed, and EMMPRIN concentrations of samples are able to determine using the calibration curve. 

Kit Components

  • Antibody pre-coated 96-well microplate --- 1 plate (6 strips)
  • Biotinylated antibody, concentrated (100 x) --- 1 vial (120 μL)
  • HRP-conjugated streptavidin, concentrated (100 x) --- 1 vial (120 μL)
  • Antibody diluent --- 1 vail (30 mL)
  • Wash solution, concentrated (20 x) --- 1 vial (30 mL)
  • Substrate solution --- 1 vial (30 mL)
  • OPD tablet --- 2 tablets
  • Stop solution (sulphuric acid) --- 1 vial (15 mL)
  • Sample diluent --- 1 vail (15 mL)
  • EMMPRIN standard (1000 ng/mL) --- 2 vial (30 μL/vial) 
Procedural Hints
  • The kit should be stored at the appropriate condition, and not be used beyond the expiry date.
  • Reagents with different lot numbers should not be mixed.
  • The thawed reagents should be stored at 2-8°C and used within 1 week.
  • The prepared (diluted) reagents should be used within the day.
  • Clinical samples should be handled as potentially infectious agents. 
  • The assay procedure should be continually performed. The wells removed aliquot should not be kept dry. 
Assay Procedure
  1. Pipet 100 μL of calibrators and samples into the wells of Antibody pre-coated 96-well microplate and incubate  at room temperature (25°C) for 1 hour.
  2. Wash the wells 3 times with 300 μL of Wash solution, and tap the plate for no droplets remaining.
  3. Pipet 100 μL of Biotinylated antibody solution into the wells and incubate at room temperature (25°C) for 1  hour. 
  4. Perform washing step about 2. 
  5. Pipet 100 μL of HRP-conjugated streptavidin solution into the wells and incubate at room temperature  (25°C) for 30 min. 
  6. Perform washing step about 2. 
  7. Pipet 100 μL of Chromogenic substrate solution into the wells and incubate at room temperature (25°C) for 10  min.
  8. Add 100 μL of Stop solution into the wells.
  9. Measure the optical density of the wells at 490 nm using a microplate reader. 

Calculation
The EMMPRIN concentrations are recommended to calculate based on the calibration curve using a built-in program of the most microplate readers.
For manual, first find the absorbance value on the Y-axis and extend a horizontal line to the calibration curve. At the point of intersection, extend a vertical line to the X-axis and read the corresponding EMMPRIN concentration.
KG573_Fig1.jpg 
Kit Performance
Sensitivity (the corresponding concentration of blank absorbance + 2SD); 0.30 ng/mL
Intra assay Precision (n = 10); CV < 10%
Inter assay Precision (n = 10); CV < 10% 

Cautions
  • For research purpose only. Not for in vitro diagnostic use.
  • Handle with care the reagents. Especially, avoid contact with sulphuric acid, substrate solution and OPD. Protect your eye, mouth, skin and clothing.
  • Follow your local regulation for disposal of all waste materials
References
  1. Yoshida S. et al. (2000) Eur J Biochem 267, 4372-4380
  2. Millimaggi D. et al. (2007) Neoplasia 9(4), 349 – 357
  3. Kathryn T. et al. (2007) Exp Mol Pathol 83(3), 283–295
  4. Reimers N. et al. (2004) Clin Cancer Res 10, 3442-3428
  5. Egawa N. et al. (2006) J Biol Chem 281(49), 37576 – 37585
  6. Tang, Y. et al. (2004) Mol Cancer Res 2, 73-80 
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